Use of Huntingtin Protein for the Diagnosis and the Treatment of Cancer

ABSTRACT

The present invention relates to new methods of treatment of cancer, in particular of breast cancer, and methods of screening of compounds useful in the treatment of cancer. The present invention further provides new prognostic and/or diagnostic markers in human cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/233,864, filed Aug. 14, 2009, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to the field of medicine, in particular ofoncology. It relates to new methods of treatment of cancer and methodsof screening of compounds useful in the treatment of cancer. The presentinvention further provides new prognostic and/or diagnostic markers inhuman cancer.

BACKGROUND OF THE INVENTION

Cancer occurs when cell division gets out of control and results fromimpairment of a DNA repair pathway, the transformation of a normal geneinto an oncogene or the malfunction of a tumor supressor gene. Manydifferent forms of cancer exist. The incidence of these cancers variesbut it represents the second highest cause of mortality, after heartdisease, in most developed countries. While different forms of cancerhave different properties, one factor which many cancers share is theability to metastasize. Distant metastasis of all malignant tumorsremains the primary cause of death in patients with the disease.

Breast cancer is a significant health problem for women in the UnitedStates and throughout the world. Although advances have been made indetection and treatment of the disease, breast cancer remains theleading cause of cancer death in women due to recurrence of local anddistant metastasis and approximately 40% of the treated patients relapseand ultimately die of metastatic breast cancer.

No universally successful method for the prevention or treatment ofbreast cancer is available and the management of the disease currentlyrelies on an early diagnosis and aggressive treatment, which may includesurgery, radiotherapy, chemotherapy and hormone therapy. However, thehigh mortality observed in breast cancer patients indicates thatimprovements are needed in the treatment, diagnosis and prevention ofthis disease.

Prostate cancer is the most common cancer and the second leading causeof cancer-related deaths among men in North America and Europe. Promptdetection and treatment is needed to limit mortality caused by prostatecancer. Localized prostate tumours are commonly diagnosed andconventionally treated by radical prostatectomy. Overwhelming clinicalevidence shows that human prostate cancer has the propensity tometastasize to bone, and the disease appears to progress inevitably fromandrogen dependent to androgen refractory status, leading to increasedpatient mortality.

In spite of considerable research into therapies for the disease,prostate cancer remains difficult to diagnose and treat effectively.Accordingly, there is a need in the art for improved methods fordetecting and treating such cancers.

SUMMARY OF THE INVENTION

The inventors surprisingly demonstrate herein that huntingtin (htt)protein, in particular the phosphorylated form of this protein, has aprotective effect against the progression of cancer.

Accordingly, in a first aspect, the present invention concerns a methodfor treating a cancer in a subject, the method comprising administeringa therapeutically effective amount of a compound increasing the cellularlevel of the phosphorylated form of huntingtin.

Preferably, the huntingtin protein is phosphorylated at one or severalpositions selected from the group consisting of S421, S535, S1181,S1201, S2076, S2653 and S2657. More preferably, the huntingtin proteinis phosphorylated at position S421.

In an embodiment, the compound increasing the cellular level of thephosphorylated form of huntingtin inhibits the dephosphorylation ofhuntingtin. Preferably, said compound is a calcineurin inhibitor or acompound inhibiting the interaction between calcineurin and huntingtin.The calcineurin inhibitor may be selected from the group consisting ofFK506, cyclosporin A, FK520, L685,818, FK523, 15-0-DeMe-FK-520, Lie120,fenvalerate, resmethrin, cypermethrin, deltamethrin and an analoguethereof. Alternatively, the calcineurin inhibitor may be a nucleic acidmolecule interfering specifically with calcineurin expression,preferably a RNAi, an antisense nucleic acid or a ribozyme. Thecalcineurin inhibitor may also be a dominant-interfering form ofcalcineurin.

In another embodiment, the compound increasing the cellular level of thephosphorylated form of huntingtin is selected from the group consistingof huntingtin protein, huntingtin protein comprising the mutation S241Dand a biologically active fragment thereof, and a nucleic acid encodingthem.

In a further embodiment, the compound increasing the cellular level ofthe phosphorylated form of huntingtin increases the phosphorylation ofhuntingtin. Preferably, said compound increases the activity of thekinase Akt, protein kinase A, Polo kinase 1, AuroraA and AuroraB and/orSGK.

Preferably, the cancer to be treated is an invasive cancer and/or acancer capable of metastasis. More preferably, the cancer is selectedfrom the group consisting of leukemia, lymphoma, melanoma, lung cancer,bowel cancer, colon cancer, rectal cancer, colorectal cancer, braincancer, liver cancer, pancreatic cancer, breast cancer, prostate cancer,testicular cancer and retinoblastoma. Even more preferably, the canceris breast cancer or prostate cancer.

In a preferred embodiment, the subject is a human. In a particularembodiment, the subject is a human not affected with Huntington'sdisease.

In a second aspect, the present invention concerns a method fordiagnosing or detecting a cancer in a subject, wherein the methodcomprises the step of determining the cellular level of phosphorylatedform of huntingtin in a sample from said subject, a low cellular levelof phosphorylated huntingtin indicating that said subject suffers from acancer. In an embodiment, the method further comprises the step ofcomparing the cellular level of phosphorylated huntingtin to a referencecellular level, preferably to the cellular level of phosphorylatedhuntingtin in a normal sample.

In a third aspect, the present invention concerns a method forpredicting, prognosing or monitoring clinical outcome of a subjectaffected with a cancer, wherein the method comprises the step ofdetermining the expression level of huntingtin in a cancer sample fromsaid subject, a low expression level of huntingtin being indicative of apoor prognosis. Preferably, the method further comprises the step ofcomparing the expression level of huntingtin to a reference expressionlevel.

In a fourth aspect, the present invention concerns a method forpredicting, prognosing or monitoring clinical outcome of a subjectaffected with a cancer, wherein the method comprises the step ofdetermining the cellular level of phosphorylated huntingtin in a cancersample from said subject, a low cellular level of phosphorylatedhuntingtin being indicative of a poor prognosis. Preferably, the methodfurther comprises the step of comparing the cellular level ofphosphorylated huntingtin to a reference cellular level.

In another aspect, the present invention concerns a method forpredicting, prognosing or monitoring clinical outcome of a subjectaffected with a cancer, wherein the method comprises the step ofdetermining the number of glutamine residues on the poly-Q expansion ofhuntingtin in a sample from said subject, a poly-Q expansion comprisingmore than 20 glutamine residues being indicative of a poor prognosis. Inan embodiment, a poly-Q expansion comprising more than 35 glutamineresidues is indicative of a poor prognosis. In another embodiment, apoly-Q expansion comprising more than 40 glutamine residues isindicative of a poor prognosis. In an embodiment, the sample from thesubject is a cancer sample.

In further aspect, the present invention concerns a method for selectinga subject affected with a cancer for an antitumoral therapy, preferablyan adjuvant chemotherapy and/or radiotherapy or determining whether asubject affected with a cancer is susceptible to benefit from anantitumoral therapy, preferably an adjuvant chemotherapy and/orradiotherapy, wherein the method comprises the step of determining thecellular level of phosphorylated huntingtin in a cancer sample from saidsubject, a low cellular level of phosphorylated huntingtin indicatingthat an antitumoral therapy, preferably an adjuvant chemotherapy and/orradiotherapy, is required.

In another aspect, the present invention concerns a method for selectinga subject affected with a cancer for an antitumoral therapy, preferablyan adjuvant chemotherapy and/or radiotherapy, or determining whether asubject affected with a cancer is susceptible to benefit from anantitumoral therapy, preferably an adjuvant chemotherapy and/orradiotherapy, wherein the method comprises the step of determining theexpression level of huntingtin in a cancer sample from said subject asdescribed above, a low expression level of huntingtin indicating that anantitumoral therapy, preferably an adjuvant chemotherapy and/orradiotherapy, is required.

In another aspect, the present invention concerns a method for selectinga subject affected with a cancer for an antitumoral therapy, preferablyan adjuvant chemotherapy and/or radiotherapy, or determining whether asubject affected with a cancer is susceptible to benefit from anantitumoral therapy, preferably an adjuvant chemotherapy and/orradiotherapy, wherein the method comprises the step of determining thenumber of glutamine residues on the poly-Q expansion of huntingtin in asample from said subject as described above, a poly-Q expansioncomprising more than 20 glutamine residues indicating that anantitumoral therapy, preferably an adjuvant chemotherapy and/orradiotherapy, is required. In an embodiment, the sample from the subjectis a cancer sample.

In a last aspect, the present invention concerns a method for selecting,identifying or screening a compound useful for treating a subject havingcancer, comprising the selection or identification of a compound capableof increasing the expression level and/or the phosphorylation ofhuntingtin.

In an embodiment, the method comprises:

a) providing a huntingtin protein or a fragment thereof of at least 50consecutive amino acids and comprising at least one phosphorylatedresidue selected from the group consisting of S421, S535, S1181, S1201,S2076, S2653 and S2657;

b) providing a compound dephosphorylating at least one phosphorylatedresidue comprised in htt protein or the fragment thereof provided instep a);

c) contacting a candidate compound with said huntingtin protein orfragment thereof and said dephosphorylating compound; and,

d) selecting the candidate compound that inhibits the dephosphorylationof at least one phosphorylated residue comprised in htt protein or thefragment thereof provided in step a) by dephosphorylating compound.

In another embodiment, the method comprises:

a) contacting a candidate compound with a cell expressing a huntingtinprotein and comprising a kinase which phosphorylates huntingtin at aposition selected from the group consisting of S421, S535, S1181, S1201,S2076, S2653 and S2657, and a compound dephosphorylating thephosphorylated residue at selected position;

b) assessing the amount of huntingtin phosphorylated and/or the amountof huntingtin which is not phosphorylated; and,

c) selecting the candidate compound that increases the phosphorylationof huntingtin at selected position in comparison with a control cellwhich has not been contacted with the candidate compound.

In a further embodiment, the method comprises:

a) contacting a candidate compound with a cell expressing a huntingtinprotein;

b) assessing the amount of huntingtin expressed in said cell; and

c) selecting the candidate compound that increases the expression ofhuntingtin in comparison with a control cell which has not beencontacted with the candidate compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1I-1J. Htt phosphorylation is lost in invasive mammary cancercells. A, Upper panel, western blot analysis of four mammary cell linesand two neuronal cells lines. Lower panel, coomassie blue staining todemonstrate total protein levels. B, Upper panel, western blot analysisof normal and tumor samples from two cases of ductal carcinomas and twocases of lobular carcinomas, showing higher levels in tumors than innormal cells. Lower panel, coomassie blue staining. C, E, G, Total Httstaining of healthy mammary lobules, an in situ tumor, and invasivecancer cells, respectively, from human biopsy samples. D, F, H,Phospho-S421-Htt staining of healthy mammary lobules, an in situ tumor,and invasive cancer cells, respectively, from the same patient as in C,E, and G (scale bar in H 20 μm). I and J, Quantification of httphosphorylation in invasive ductal and lobular cancers, respectively,from human biopsy samples. Only samples that contained positivelystained residual healthy structures were included in the analysis.

FIGS. 2A-2E. Htt regulates cell motility. A, Confocal images of 2a1cells expressing shLuc (upper), shHtt (middle), or shHtt plus anshRNA-resistant CTFL-Htt. The shRNA constructs express GFP from aseparate promoter, allowing identification of transfected cells. Theright column shows that the addition of CTFL-Htt rescues theshHtt-induced decrease in total htt to the endogenous levels inshLuc-expressing cells (compare lower right panel to upper right panel).B and C, Scratch closure assays in MCF7 and 4T1 cells, respectively,showing faster closure in cells that were depleted of endogenous httthan in controls. D, Random migration assays of shLuc- andshHtt-expressing MCF7 cells. E, Random migration assays of 2a1transfected with shLuc+cherry, shHtt+cherry, shHtt+CTFLHtt-WT,shHtt+CTFLHtt-S421A, and shHtt+CTFLHtt-S421D, showing that theunsphosphorylatable S421A mutation does not rescue the increasedvelocity induced by the depletion of endogenous htt, while the S421-WTand S421D forms do.

FIGS. 3A-3E. Htt controls metastasis and invasion. A, Western blotanalysis showing the efficiency of siHtt in 4T1 cells in vitro. B and C,Bioluminescence images merged to photos of Balb/C mice injected with 4t1cells and treated with seven days of either a scramble RNA (sc) or siHtt(quantified in C, n=11 per group, p<0.01). D, Survival curve of the samesc- and siHtt-treated mice from B and C, showing a significant decreasein the survival of siHtt-treated mice by Kaplan-Meier analysis(p=0.012). E, Images of the lungs from sc- (top) and siHtt- (bottom)treated mice. The three left-most columns show (from left to right) theluciferase, total htt, and nuclear images, obtained by confocalmicroscopy. The right-most column shows images of whole lungs dissectedfrom mice at the time of death. F, Matrigel Invasion analysis of 4T1cells stably expressing sc or siHtt without or with different CTFLHttconstructs. The siHtt+CTFLS421A cells showed significantly higherinvasion (p<0.001 for all cases) than any of the other groups. No othersignificant differences were observed.

FIGS. 4A-4B. Oncogene-induced mammary tumors develop faster in HD mousemodel. (A) MMTV-PyVT and MMTV-ErbB2 mice were crossed with theHdh^(Q111/Q111) mouse line carrying a 111 CAG expansion inserted intothe endogenous mouse huntingtin gene. Tumor appearance was followed as afunction of time (days). WT: MMTV-ErbB2/PyVT; Hdh^(Q7/Q7); Q111/Q111:MMTV-ErbB2/PyVT; Hdh^(Q111/Q111). (B) Whole mount of mammary glands werestained with carmine aluminium. Images were acquired with a stereomicroscope (0.63×).

FIG. 5. PolyQ-huntingtin does not change tumor growth. Primarysolid-tumor isolates from MMTV-PyVT; Hdh^(Q111/Q111) and MMTV-PyVT;Hdh^(Q7/Q7) (WT) were transplanted in immunodeficient mice and tumorsize was measured as a function of time following transplantation(days).

FIGS. 6A-6C. PolyQ-huntingtin leads to increased cell motility,invasiveness and metastasis. (A) Random cell migration and (B) matrigelassays were performed with primary cells derived from MMTV-pyVT;Hdh^(Q7/Q7) and MMTV-PyVT; Hdh^(Q111/Q111) tumors. (C) Lungs fromimmunodeficient mice grafted with primary solid-tumor isolates fromMMTV-ErbB2; Hdh^(Q111/Q111) and MMTV-ErbB2; Hdh^(Q7/Q7) were dissected,sectioned and stained with hematoxylin and eosin.

FIG. 7. Breast cancer onset is correlated with the CAG length in HDpatients. Nine HD patients developing breast cancer were identified. Thelength of their abnormal CAG expansion is represented as a function oftheir age of HD and breast cancer onsets.

DETAILED DESCRIPTION OF THE INVENTION

Huntingtin (htt) is a large protein whose function and regulation havenot been well defined. Expansion of a polyglutamine repeat within thehuntingtin protein is known to be the causative event of Huntington'sdisease (HD), a progressive neurological disorder that leads to adistinctive chorea, cognitive loss, various psychological disorders, andeventually death. As the mutation that causes HD has been known foralmost 15 years, the majority of researches have focused on htt duringdisease progression instead of on the role of the wild-type htt protein.As a consequence, very few elements are known about the role ofwild-type htt.

Studies on the cellular localization of the mRNA and protein haverevealed that wild-type and polyQ-huntingtin are expressed throughoutthe nervous system as well as in non-neuronal cells (Sharp and Ross,1996) suggesting that the wild-type protein's main function may notnecessarily be brain-specific.

Some other studies demonstrated an ambiguous correlation between htt andcancer. In 1999, it was suggested that the lower incidence of canceramong HD patients could be due to the polyQ-huntingtin, encountered inHD, which would be able to protect HD patient against cancer by inducingor increasing the rate of naturally occurring programmed cell death inpreneoplastic cells (Sorensen et al., 1999). This theory was furthersupported by two studies demonstrating that the polyglutamine expansionis responsible for cell death induction and that this cell death ismediated by caspase (Saudou et al., 1998; Martindale et al., 1998; Wanget al., 1999). In addition, it was also demonstrated that wild-type httmay be an anti-apoptotic protein (Zeitlin et al., 1995; Rigamonti etal., 2000; Leavitt et al., 2001) and the US patent application US2002-0187931 describes the use of antagonists to huntingtin protein totreat conditions characterized by dysregulation of cellularproliferation such as cancer.

On the contrary, the inventors herein surprisingly show that wild-typehtt, and in particular the phosphorylated form of htt, can beefficiently used in the treatment of cancer. As described in theexperimental section, the inventors demonstrate that the decrease ofcellular level of the phosphorylated form of htt is associated with theincrease of cell motility, invasive and metastasis capacities of cancercells. Furthermore, they show that the administration of htt or of thephosphorylated form of htt completely rescues the increased motilityinduced by the knock-down of endogenous htt.

Definitions

As used herein, the term “huntingtin”, “huntingtin protein”, or “htt”refers to the huntingtin protein encoded by the huntingtin gene alsocalled HTT gene, HD gene or IT15 gene (GeneID: 3064). There are manypolymorphisms of this gene due to a variable number of CAG codonrepeats, encoding glutamine, in the first exon. In its wild-type form,i.e. htt form not causing Huntington's disease, this protein containsfrom 6 to 35 glutamine residues. In individuals affected by HD, thisprotein contains more than 35 glutamine residues and is named polyQ-htt.The NCBI accession number of wild-type htt is NP_(—)002102 (SEQ ID NO:1). The wild-type form of htt has a mass about 350 kD and a size ofabout 3144 amino acids. Unless otherwise specified, the term “htt”, asused herein, refers to the wild-type form of the htt protein, i.e. httcontaining a polyglutamine tract of less than 36 glutamine residues. Httmay be phosphorylated at one or several positions selected from thegroup consisting of S421, S535, S1181, S1201, S2076, S2653 and S2657.These positions refer to the positions of amino acids in the NCBIsequence of wild-type htt with the accession number NP_(—)002102(sequence of SEQ ID NO: 1).

The term “cancer” or “tumor”, as used herein, refers to the presence ofcells possessing characteristics typical of cancer-causing cells, suchas uncontrolled proliferation, immortality, metastatic potential, rapidgrowth and proliferation rate, and certain characteristic morphologicalfeatures. This term refers to any type of malignancy (primary ormetastases). Typical cancers are solid or hematopoietic cancers such asbreast, stomach, oesophageal, sarcoma, ovarian, endometrium, bladder,cervix uteri, rectum, colon, lung or ORL cancers, paediatric tumours(neuroblastoma, glioblastoma multiforme), lymphoma, leukaemia, myeloma,seminoma, Hodgkin and malignant hemopathies. Preferably, the cancer tobe treated is an invasive cancer and/or a cancer capable of metastasis.In a particular embodiment, the cancer is selected from the groupconsisting of leukemia, lymphoma, melanoma, lung cancer, bowel cancer,colon cancer, rectal cancer, colorectal cancer, brain cancer, livercancer, pancreatic cancer, breast cancer, prostate cancer, testicularcancer and retinoblastoma. In a preferred embodiment, the cancer is asolid cancer, preferably a breast cancer or a prostate cancer, morepreferably a breast cancer. Preferably the cancer is an early stagecancer without local or systemic invasion, more preferably an earlystage breast cancer without local or systemic invasion.

As used herein, the term “treatment of cancer” refers to any actintended to extend life span of patients such as therapy and retardationof the disease. The treatment can be designed to eradicate the tumor, tostop the progression of the tumor, to prevent the occurrence ofmetastasis, to promote the regression of the tumor and/or to preventmuscle invasion of cancer. Preferably, the term “treatment of cancer” asused herein, refers to the prevention or delay of metastasis formation,disease progression and/or muscle invasion. The term “therapy” or“antitumoral therapy”, as used herein, refers to any type of treatmentof cancer, including an adjuvant therapy and a neoadjuvant therapy.Therapy comprises radiotherapy and therapies, preferably systemictherapies such as hormone therapy, chemotherapy, immunotherapy andmonoclonal antibody therapy.

As used herein, the term “chemotherapy” refers to a cancer therapeutictreatment using chemical or biochemical substances, in particular usingone or several antineoplastic agents.

The term “radiotherapy” is a term commonly used in the art to refer tomultiple types of radiation therapy including internal and externalradiation therapies or radioimmunotherapy, and the use of various typesof radiations including X-rays, gamma rays, alpha particles, betaparticles, photons, electrons, neutrons, radioisotopes, and other formsof ionizing radiations.

The term “immunotherapy” refers to a cancer therapeutic treatment usingthe immune system to reject cancer. The therapeutic treatment stimulatesthe patient's immune system to attack the malignant tumor cells. Itincludes immunization of the patient with tumoral antigens (e.g. byadministering a cancer vaccine), in which case the patient's own immunesystem is trained to recognize tumor cells as targets to be destroyed,or administration of molecules stimulating the immune system such ascytokines, or administration of therapeutic antibodies as drugs, inwhich case the patient's immune system is recruited to destroy tumorcells by the therapeutic antibodies. In particular, antibodies aredirected against specific antigens such as the unusual antigens that arepresented on the surfaces of tumors. As illustrating example, one cancite Trastuzumab or Herceptin antibody which is directed against HER2and approved by FDA for treating breast cancer.

The term “monoclonal antibody therapy” refers to any antibody thatfunctions to deplete tumor cells in a patient. In particular,therapeutic antibodies specifically bind to antigens present on thesurface of the tumor cells, e.g. tumor specific antigens presentpredominantly or exclusively on tumor cells. Alternatively, therapeuticantibodies may also prevent tumor growth by blocking specific cellreceptors.

The term “hormone therapy” or “hormonal therapy” refers to a cancertreatment having for purpose to block, add or remove hormones. Forinstance, in breast cancer, the female hormones estrogen andprogesterone can promote the growth of some breast cancer cells. So inthese patients, hormone therapy is given to block estrogen and anon-exhaustive list commonly used drugs includes: Tamoxifen, Fareston,Arimidex, Aromasin, Femara, Zoladex/Lupron, Megace, and Halotestin.

The term “adjuvant therapy”, as used herein, refers to any type oftreatment of cancer (e.g., chemotherapy or radiotherapy) given asadditional treatment, usually after surgical resection of the primarytumor, in a patient affected with a cancer that is at risk ofmetastasizing and/or likely to recur. The aim of such an adjuvanttreatment is to improve the prognosis. Adjuvant therapies compriseradiotherapy and therapy, preferably systemic therapy, such as hormonetherapy, chemotherapy, immunotherapy and monoclonal antibody therapy.

The term “neoadjuvant therapy”, as used herein, refers to any type oftreatment of cancer given prior to surgical resection of the primarytumor, in a patient affected with a cancer. The most common reason forneoadjuvant therapy is to reduce the size of the tumor so as tofacilitate a more effective surgery. Neoadjuvant therapies compriseradiotherapy and therapy, preferably systemic therapy, such as hormonetherapy, chemotherapy, immunotherapy and monoclonal antibody therapy.

The term “therapeutically effective amount” refers to that amount of atherapy which is sufficient to reduce or ameliorate the severity,duration and/or progression of a disease or one or more symptomsthereof. As used herein, this term refers to that amount of a compoundincreasing the cellular level of the phosphorylated form of huntingtin,which is sufficient to destroy, modify, control or remove primary,regional or metastatic cancer tissue, ameliorate cancer or one or moresymptoms thereof, or prevent the advancement of cancer, cause regressionof cancer, or enhance or improve the therapeutic effect (s) of anothertherapy (e.g., a therapeutic agent). This term may also refer to theamount of a compound of the present invention sufficient to delay orminimize the spread of cancer or sufficient to provide a therapeuticbenefit in the treatment or management of cancer. Further, atherapeutically effective amount with respect to a compound of thepresent invention means that amount of a compound of the presentinvention alone, or in combination with other therapeutic agent, thatprovides a therapeutic benefit in the treatment or management of cancer.

As used herein, the term “poor prognosis” refers to a decreased patientsurvival and/or an early disease progression and/or an increased cancerinvasion and/or an increased metastasis formation.

As used herein, the term “subject” or “patient” refers to an animal,preferably to a mammal, even more preferably to a human, includingadult, child and human at the prenatal stage. However, the term“subject” can also refer to non-human animals, in particular mammalssuch as dogs, cats, horses, cows, pigs, sheep and non-human primates,among others, that are in need of treatment.

The term “sample”, as used herein, means any sample containing cellsderived from a subject. Examples of such samples include fluids such asblood, plasma, saliva, urine and seminal fluid samples as well asbiopsies, organs, tissues or cell samples. The sample may be treatedprior to its use. The term “cancer sample” refers to any samplecontaining tumoral cells derived from a patient. The term “normalsample” refers to any sample which does not contain any tumoral cells.

The methods of the invention as disclosed below, may be in vivo, ex vivoor in vitro methods, preferably in vitro methods.

As demonstrated in the experimental section, the inventors show thathuntingtin (htt) protein, in particular the phosphorylated form of thisprotein, is involved in the progression of cancer and that an increaseof the cellular level of the phosphorylated form of htt allows toprevent cancer invasion and metastasis.

Accordingly, in a first aspect, the present invention concerns a methodfor treating cancer in a subject, comprising administering atherapeutically effective amount of a compound increasing the cellularlevel of the phosphorylated form of huntingtin. Said compound mayinhibit the dephosphorylation of htt, increase the phosphorylation ofhtt or increase the cellular level of wild-type htt.

In particular, the phosphorylated htt may be phosphorylated at one orseveral positions selected from the group consisting of S421, S535,S1181, S1201, S2076, S2653 and S2657. Preferably, the phosphorylated httis phosphorylated at least at position S421.

In a first embodiment, the compound of the invention inhibits thedephosphorylation of htt. Preferably, the compound inhibits thedephosphorylation of htt at position S421. More preferably, the compoundis a calcineurin inhibitor or a compound inhibiting the interactionbetween calcineurin and huntingtin.

In a preferred embodiment, the compound inhibiting the dephosphorylationof htt is a calcineurin inhibitor. Calcineurin inhibitors include, butare not limited to, cyclosporin A (Novartis International AG,Switzerland), FK506 (Fujisawa Healthcare, Inc., Deerfield, Ill., USA),FK520 (Merck & Co, Rathway, N.J., USA), L685,818 (Merck & Co), FK523,15-0-DeMe-FK-520 (Liu, Biochemistry, 31:3896-3902 (1992)), Lie120,fenvalerate (Merck & Co), resmethrin (Merck & Co), cypermethrin (Merck &Co) and deltamethrin (Merck & Co), and analogues thereof. WO2005087798describes cyclosporine derivative inhibiting calcineurin. In aparticularly preferred embodiment, the compound inhibiting thedephosphorylation of htt is selected from FK506, cypermethrin,deltamethrin and an analogue thereof. The term “analogue”, as usedherein, refers to a compound having similar structural features andhaving the same biological activity, in particular which inhibitscalcineurin.

Calcineurin is a heterodimer composed of a catalytic subunit(Calcineurin A, CaNA) and a regulator subunit (Calcineurin B, CaNB).Then, the activity of calcineurin can also be inhibited by blocking itsexpression, in particular the expression of one of its subunit. In anembodiment, the activity of calcineurin is inhibited by blocking theexpression of the catalytic subunit A, the α (GeneID: 5530), β (GeneID:5532) or γ (GeneID: 5533) isoform. In another embodiment, the activityof calcineurin is inhibited by blocking the expression of the regulatorsubunit B, either the α isofom (GeneID: 5534) or the β isoform (GeneID:5535) or both. The expression of calcineurin can be blocked by any meanknown by one skilled in the art.

In a particular embodiment, the calcineurin inhibitor is a nucleic acidmolecule interfering specifically with calcineurin expression.Preferably, this nucleic acid is selected from the group consisting of aRNAi, an antisense nucleic acid or a ribozyme.

The term “RNAi” or “interfering RNA” means any RNA which is capable ofdown-regulating the expression of the targeted protein. It encompassessmall interfering RNA (siRNA), double-stranded RNA (dsRNA),single-stranded RNA (ssRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules. RNA interference, designate a phenomenon by whichdsRNA specifically suppresses expression of a target gene atpost-translational level. In normal conditions, RNA interference isinitiated by double-stranded RNA molecules (dsRNA) of several thousandsof base pair length. In vivo, dsRNA introduced into a cell is cleavedinto a mixture of short dsRNA molecules called siRNA. The enzyme thatcatalyzes the cleavage, Dicer, is an endo-RNase that contains RNase IIIdomains (Bernstein et al. 2001). In mammalian cells, the siRNAs producedby Dicer are 21-23 bp in length, with a 19 or 20 nucleotides duplexsequence, two-nucleotide 3′ overhangs and 5′-triphosphate extremities(Zamore et al. 2000; Elbashir et al. 2001). A number of patents andpatent applications have described, in general terms, the use of siRNAmolecules to inhibit gene expression, for example, WO 99/32619, US20040053876, US 20040102408 and WO 2004/007718.

siRNA are usually designed against a region 50-100 nucleotidesdownstream the translation initiator codon, whereas 5′UTR (untranslatedregion) and 3′UTR are usually avoided. The chosen siRNA target sequenceshould be subjected to a BLAST search against EST database to ensurethat the only desired gene is targeted. Various products arecommercially available to aid in the preparation and use of siRNA. In apreferred embodiment, the RNAi molecule is a siRNA of at least about15-50 nucleotides in length, preferably about 20-30 base nucleotides.For instance, convenient siRNA nucleotides may present the sequences ofSEQ ID NOs: 2 and 3.

RNAi can comprise naturally occurring RNA, synthetic RNA, orrecombinantly produced RNA, as well as altered RNA that differs fromnaturally-occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end of the moleculeor to one or more internal nucleotides of the RNAi, includingmodifications that make the RNAi resistant to nuclease digestion.

RNAi may be administered in free (naked) form or by the use of deliverysystems that enhance stability and/or targeting, e.g., liposomes, orincorporated into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, bioadhesive microspheres, or proteinaceousvectors (WO 00/53722), or in combination with a cationic peptide (US2007275923). They may also be administered in the form of theirprecursors or encoding DNAs.

Antisense nucleic acid can also be used to down-regulate the expressionof the calcineurin. The antisense nucleic acid can be complementary toall or part of a sense nucleic acid encoding a calcineurin subunit e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence, and it thought to interfere with thetranslation of the target mRNA; Preferably, the antisense nucleic acidis a RNA molecule complementary to a target mRNA encoding a calcineurinsubunit.

An antisense nucleic acid can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. Particularly, antisense RNAmolecules are usually 18-50 nucleotides in length.

An antisense nucleic acid can be constructed using chemical synthesisand enzymatic ligation reactions using procedures known in the art.Particularly, antisense RNA can be chemically synthesized, produced byin vitro transcription from linear (e.g. PCR products) or circulartemplates (e.g., viral or non-viral vectors), or produced by in vivotranscription from viral or non-viral vectors.

Antisense nucleic acid may be modified to have enhanced stability,nuclease resistance, target specificity and improved pharmacologicalproperties. For example, antisense nucleic acid may include modifiednucleotides designed to increase the physical stability of the duplexformed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides.

Ribozyme molecules can also be used to block the expression of acalcineurin subunit. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes can be used to catalytically cleave mRNAtranscripts to thereby inhibit translation of the protein encoded by themRNA. Ribozyme molecules specific for a calcineurin subunit can bedesigned, produced, and administered by methods commonly known to theart (see e.g., Fanning and Symonds, 2006, reviewing therapeutic use ofhammerhead ribozymes and small hairpin RNA).

In a particular embodiment, the interfering nucleic acid molecule isexpressed by a vector, preferably a viral vector comprising a contructallowing the expression of interfering nucleic acid molecule. Forinstance, the viral vector can be an adenovirus, an adeno-associatedvirus, a lentivirus or a herpes simplex virus.

Calcineurin inhibitor can also be a dominant-interfering form ofcalcineurin, in particular of CaNA and/or of CaNB. Thedominant-interfering form of calcineurin can be for example CaNA-D130N.Other examples of dominant-interfering form of calcineurin are

CaNA-H101Q (Wang et al, 1999, Science 284, 339-343), CaNA-H160Q andCaNA-H290Q (Shibasaki et al, 1996, Nature, 6589, 370-373; Nishimura andTanaka, 2001, J. Biol. Chem., 276, 19921-19928), H160Q (Zhu et al, 2000nJ. Biol. Chem., 275, 15239-15245), D148-152 (Yamashita, 2000, J. Exp.Med., 191, 1869-1880) and Dnter-DcaM (muramatsu and Kincaid, 1996, BBRC,218, 466-472; Musaro et al, 1999, Nature, 6744, 581-585). Therefore, thedominant-interfering form of calcineurin is expressed by a vector,preferably a viral vector comprising a contruct allowing its expression.For instance, the viral vector can be an adenovirus, an adeno-associatedvirus, a lentivirus or a herpes simplex virus.

Alternatively, the activity of calcineurin can be inhibited by a drugthat inhibits the interaction between the calcineurin subunits, inparticular the interaction between subunits A and B. The activity ofcalcineurin can be inhibited by a drug that inhibits the interactionbetween the calcineurin and calmodulin. Calcineurin inhibition can alsobe obtained by activation of endogenous inhibitors of calcineurin,including cabin1, calcipressins and AKAP79.

Other drugs inhibiting the calcineurin can be identified by screeningmethods already disclosed in the art. As illustration, the U.S. Pat.Nos. 6,875,581 and 6,338,946 describe screening methods useful foridentifying modulators of calcineurin activity.

In another embodiment, the compound of the invention increases thephosphorylation of htt. Preferably, the compound increases thephosphorylation of htt at position S421. This compound may increase theactivity of a kinase phosphorylating htt. In a particular embodiment,the compound increases the activity of a kinase selected from the groupof protein kinase A, Polo kinase 1, cyclin-dependent kinase 5 (Cdk5),kinase Akt, AuroraA and AuroraB and/or SGK. Preferably, the compoundincreases the activity of a kinase selected from the group of proteinkinase A, Polo kinase 1, cyclin-dependent kinase 5 (Cdk5),cyclin-dependent kinase 1 (Cdk1), cyclin-dependent kinase 2 (Cdk2),AuroraA and AuroraB and/or SGK. The compound increasing thephosphorylation of htt may be a nucleic acid encoding a protein kinaseinvolved in the phosphorylation of htt. This compound may also increasethe cellular level of a kinase phosphorylating htt by increasing theexpression level of the endogenous kinase.

In a further embodiment, the compound of the invention increases thecellular level of phosphorylated htt by increasing the cellular level ofwild-type htt. Said compound may be selected from the group consistingof huntingtin protein and a biologically active fragment thereof,huntingtin protein comprising the mutation S241D and a biologicallyactive fragment thereof, and a nucleic acid encoding thereof.

In an embodiment, the compound of the invention is wild-type htt.

In another embodiment, the compound of the invention is a biologicallyactive fragment of htt protein. In a particular embodiment, thebiologically active fragment of htt is a N-terminal fragment of thewild-type htt protein comprising at least 500 residues. This fragmentmay be obtained, for example, by proteolysis of htt by caspase-3 orcaspase-6. The term “biologically active fragment of huntingtinprotein”, as used herein, refers to a fragment of htt which is able tomaintain a normal cell motility. As the inventors have demonstrated thatthe knock-down of endogenous htt increases cell motility, the term“biologically active fragment of huntingtin protein” refers to afragment of htt which is able to rescue the increased motility inducedby this knock-down. This capacity may be assessed in a cellco-transfected with a RNA construct blocking the expression of htt, e.g.a shRNA htt, and with a nucleic acid encoding the htt fragment to betested. The motility of cell is then measured and compared to themotility of a control cell transfected only with a RNA constructblocking the expression of endogenous htt. A reduced cell motilitycompared to the motility of the control cell indicates that the httfragment encoded by the nucleic acid is biologically active and can beused in the present invention. This test is further detailed in theexperimental section.

In a preferred embodiment, the compound of the invention is huntingtinprotein comprising the mutation S421D. This protein has been previouslydescribed in Humbert et al. (Humbert et al., 2002). The mutation S421Dmimics constitutive phosphorylation of htt. In a particular embodiment,the compound of the invention is a biologically active fragment ofhuntingtin protein comprising the mutation S421D, preferably aN-terminal fragment of said htt protein comprising at least 500residues. The activity of said fragment may be assessed as describedabove.

Polypeptides, including htt proteins and their fragments, used in thepresent invention may be recombinant, purified or isolated polypeptides.The terms “recombinant polypeptide” is used herein to refer topolypeptides that have been artificially designed. A recombinantpolypeptide is usually a polypeptide which has been expressed from arecombinant nucleic acid molecule. The term “purified polypeptide” isused herein to describe a polypeptide which has been separated fromother compounds including, but not limited to, lipids, carbohydrates,nucleic acids and other proteins. A polypeptide is substantially purewhen at least about 50%, preferably 60 to 75%, more preferably 80 to95%, and even more preferably 95 to 99% of a sample exhibits a singlepolypeptide sequence. Polypeptide purity or homogeneity may be verifiedby a number of means well known in the art, such as polyacrylamide gelelectrophoresis. The term “isolated” requires that the material isremoved from its original environment. The term “isolated polypeptide”refers to a polypeptide that is separated from some or all of thecoexisting materials in its natural environment and preferablysubstantially free from any other contaminating polypeptides which wouldinterfere with its therapeutic use.

In a particular embodiment, the compound increasing the cellular levelof wild-type htt is a nucleic acid encoding htt protein or htt proteincomprising the mutation. S421D or any biologically active fragmentthereof. The nucleic acid may be inserted in a vector allowing itsexpression in eukaryote cells. The structure and composition of suchvectors is well-known by the skilled person. Preferably, the vector is aviral vector such as an adenovirus, an adeno-associated virus, alentivirus or a herpes simplex virus.

The pharmaceutical composition comprising a compound increasing thecellular level of the phosphorylated form of htt is formulated inaccordance with standard pharmaceutical practice (see, e.g., Remington:The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro,Lippincott Williams & Wilkins, 2000 and Encyclopedia of PharmaceuticalTechnology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, MarcelDekker, New York) known by a person skilled in the art.

Possible pharmaceutical compositions include those suitable for oral,rectal, topical (including transdermal, buccal and sublingual),intratumoral or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. For these formulations,conventional excipient can be used according to techniques well known bythose skilled in the art.

The compositions for parenteral administration are generallyphysiologically compatible sterile solutions or suspensions which canoptionally be prepared immediately before use from solid or lyophilizedform. Adjuvants such as a local anesthetic, preservative and bufferingagents can be dissolved in the vehicle and a surfactant or wetting agentcan be included in the composition to facilitate uniform distribution ofthe active ingredient.

For oral administration, the composition can be formulated intoconventional oral dosage forms such as tablets, capsules, powders,granules and liquid preparations such as syrups, elixirs, andconcentrated drops. Non toxic solid carriers or diluents may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose,glucose, sucrose, magnesium, carbonate, and the like. For compressedtablets, binders, which are agents which impart cohesive qualities topowdered materials are also necessary. For example, starch, gelatine,sugars such as lactose or dextrose, and natural or synthetic gums can beused as binders. Disintegrants are also necessary in the tablets tofacilitate break-up of the tablet. Disintegrants include starches,clays, celluloses, algins, gums and crosslinked polymers. Moreover,lubricants and glidants are also included in the tablets to preventadhesion to the tablet material to surfaces in the manufacturing processand to improve the flow characteristics of the powder material duringmanufacture. Colloidal silicon dioxide is most commonly used as aglidant and compounds such as talc or stearic acids are most commonlyused as lubricants.

For transdermal administration, the composition can be formulated intoointment, cream or gel form and appropriate penetrants or detergentscould be used to facilitate permeation, such as dimethyl sulfoxide,dimethyl acetamide and dimethylformamide.

For transmucosal administration, nasal sprays, rectal or vaginalsuppositories can be used. The active compound can be incorporated intoany of the known suppository bases by methods known in the art. Examplesof such bases include cocoa butter, polyethylene glycols (carbowaxes),polyethylene sorbitan monostearate, and mixtures of these with othercompatible materials to modify the melting point or dissolution rate.

Pharmaceutical compositions according to the invention may be formulatedto release the active drug substantially immediately upon administrationor at any predetermined time or time period after administration.

Pharmaceutical compositions according to the invention can comprise oneor more compounds increasing the cellular level of the phosphorylatedform of htt, associated with pharmaceutically acceptable excipientsand/or carriers. These excipients and/or carriers are chosen accordingto the form of administration as described above.

In a particular embodiment, the pharmaceutical composition comprises oneor more compounds increasing the cellular level of the phosphorylatedform of htt, selected from the group consisting of a compound inhibitingthe dephosphorylation of htt, a compound increasing the phosphorylationof htt and a compound increasing the cellular level of htt.

In another particular embodiment, the pharmaceutical compositioncomprises one or more compounds increasing the cellular level of thephosphorylated form of htt, selected from the group consisting of acompound inhibiting the dephosphorylation of htt and a compoundincreasing the cellular level of htt. Preferably, the pharmaceuticalcomposition comprises a compound inhibiting the dephosphorylation of httand a compound increasing the cellular level of htt. In a particularembodiment, the pharmaceutical composition comprises a calcineurininhibitor and a compound selected from the group consisting ofhuntingtin protein and a biologically active fragment thereof,huntingtin protein comprising the mutation S241D and a biologicallyactive fragment thereof, and a nucleic acid encoding thereof.

Other active molecules can also be associated with the compound of theinvention such as other molecules used for the treatment of cancer, inparticular antitumoral drugs such as tamoxifen, aromatase inhibitors,trastuzumab, GnRH-analogues, gemcitabine, docetaxel, paclitaxel,mitomycin, cisplatin, carboplatin, oxaliplatin, doxorubicin,daunorubicin, docetaxel, cyclophosphamide, epirubicin, fluorouracil,methotrexate, mitozantrone, vinblastine, vincristine, vinorelbine,bleomycin, estramustine phosphate or etoposide phosphate.

The amount of compound increasing the cellular level of thephosphorylated form of htt, to be administered has to be determined bystandard procedure well known by those of ordinary skill in the art.Physiological data of the patient (e.g. age, size, and weight), theroutes of administration and the type of cancer to be treated have to betaken into account to determine the appropriate dosage.

The compound of the invention may be administered as a single dose or inmultiple doses.

In an embodiment, the compound of the invention is a calcineurininhibitor selected from the group consisting of FK506, cyclosporin A,FK520, L685,818, FK523, 15-0-DeMe-FK-520, Lie120, fenvalerate,resmethrin, cypermethrin, deltamethrin and an analogue thereof and thedose can be, for instance, from 0.001 mg/kg/day to 10 mg/kg/day,preferably between 0.01 and 10 mg/kg/day by oral administration andbetween 0.001 and 1 mg/kg/day by intravenous injection, preferablybetween 0.01 and 0.5 mg/kg/day. The unitary dose may be, for instance,from 0.02 mg to 700 mg, preferably from 0.2 mg to 700 mg, for oraladministration and from 0.02 mg to 70 mg, preferably from 0.2 mg to 35mg, for intravenous injection. In a particular embodiment, thecalcineurin inhibitor is FK506 and the administered dose of FK506 can beadapted in order to obtain a blood FK506 level comprised between 5 and40 ng/ml, preferably between 15 and 20 ng/ml.

In another embodiment, the compound of the invention is a calcineurininhibitor is a nucleic acid molecule interfering specifically withcalcineurin expression, such as a RNAi, an antisense nucleic acid or aribozyme, and the dose can be, for instance, from 1 μg/kg/day to 10mg/kg/day. The unitary dose may be, for instance, from 70 μg to 700 mg.

In another embodiment, the compound of the invention is a calcineurininhibitor is a dominant-interfering form of calcineurin and the dose canbe, for instance, from 1 μg/kg/day to 10 mg/kg/day. The unitary dose maybe, for instance, from 70 μg to 700 mg.

If the compound of the invention is selected from the group consistingof huntingtin protein and a biologically active fragment thereof,huntingtin protein comprising the mutation S241D and a biologicallyactive fragment thereof, and a nucleic acid encoding thereof, the dosecan be, for instance, from 1 μg/kg/day to 10 mg/kg/day. The unitary dosemay be, for instance, from 70 μg to 700 mg.

In a further embodiment, the compound of the invention is a compoundincreasing the activity of the kinase Akt, protein kinase A, Polo kinase1, AuroraA and AuroraB and/or SGK, and the dose can be, for instance,from 1 μg/kg/day to 10 mg/kg/day. The unitary dose may be, for instance,from 70 μg to 700 mg.

The compound of the invention can be used in combination with otheractive ingredients, in particular with other molecules used for thetreatment of cancer. In this case, the compound of the invention and theother molecules can be administered simultaneously or consecutively.

The treatment of cancer with pharmaceutical composition according to theinvention can be associated with other therapy such as surgery,radiation therapy or other chemotherapy.

The subject to treat is any mammal, preferably a human being. In aparticular embodiment, the subject is a human being not affected by HD.

The present invention also concerns a method for preventing metastasisoccurrence and/or cancer invasion in a subject affected with a cancer,the method comprising administering a therapeutically effective amountof a compound increasing the cellular level of the phosphorylated formof huntingtin.

In an embodiment, the compound increasing the cellular level of thephosphorylated form of huntingtin is used to prevent metastasisoccurrence in a subject affected with a cancer.

In another embodiment, the compound increasing the cellular level of thephosphorylated form of huntingtin is used to prevent cancer invasion ina subject affected with a cancer.

The term “to prevent metastasis occurrence and/or cancer invasion”, asused herein, refers to delay, minimize or inhibit the spread of cancerthrough metastasis and/or invasion of surrounding tissues.

The present invention also concerns the use of a compound increasing thecellular level of the phosphorylated form of htt for the manufacture ofa medicament for treating cancer.

The present invention further concerns the use of a compound increasingthe cellular level of the phosphorylated form of htt for the manufactureof a medicament for preventing metastasis occurrence and/or cancerinvasion in a subject affected with a cancer.

The present invention further concerns a method for treating a cancer ina subject, said method comprising the administration of atherapeutically effective amount of a compound increasing the cellularlevel of the phosphorylated form of huntingtin to said subject. Suchcompound may inhibit the dephosphorylation of htt, increase thephosphorylation of htt or increase the cellular level of htt, asdescribed above.

In an embodiment, the phosphorylated htt may be phosphorylated at one orseveral positions selected from the group consisting of S421, S535,S1181, S1201, S2076, S2653 and S2657. Preferably, htt is phosphorylatedat position S421.

In a preferred embodiment, the cancer to be treated is an invasivecancer and/or a cancer capable of metastasis. The cancer may be selectedfrom the group consisting of leukemia, lymphoma, melanoma, lung cancer,bowel cancer, colon cancer, rectal cancer, colorectal cancer, braincancer, liver cancer, pancreatic cancer, breast cancer, prostate cancer,testicular cancer and retinoblastoma. Preferably, the cancer is a breastcancer or a prostate cancer, more preferably a breast cancer.

In a particular embodiment, the method further comprises the step ofassessing the cellular level of phosphorylated form of htt in a cancersample from the subject. The cellular level of phosphorylated form ofhtt may be determined by any method known by the skilled person, such asmethod as described below.

The present invention further concerns a method for preventingmetastasis occurence and/or cancer invasion in a subject affected with acancer, said method comprising the administration of a therapeuticallyeffective amount of a compound increasing the cellular level of thephosphorylated form of huntingtin to said subject.

The invention also concerns a method for decreasing the aggressivity ofa cancer in a subject, said method comprising the administration of atherapeutically effective amount of a compound increasing the cellularlevel of the phosphorylated form of huntingtin to said subject.

The inventors herein demonstrate that htt is phosphorylated in normaltissues and that this phosphorylation is lost in cancer cells, in agreater extent in invasive cancer cells. The phosphorylation state ofhtt thus constitutes a marker for diagnosis of multiple types of cancer,in particular breast cancer.

Accordingly, in another aspect, the present invention concerns a methodfor diagnosing or detecting a cancer in a subject, wherein the methodcomprises the step of determining the cellular level of phosphorylatedform of htt in a sample from said subject, a low cellular level ofphosphorylated htt indicating that said subject suffers from a cancer.

In an embodiment, the method further comprises the step of providing asample from the subject. Preferably, this sample is suspected to containtumoral cells.

The cellular level of phosphorylated form of htt may be determined byany method known by the skilled person. For instance, the cellular levelof phosphorylated form of htt may be determined by immunohistochemicalstaining of the sample with an antibody specific to the phosphorylatedform of htt, such as the anti-phospho-huntingtin-S421-763 described inthe article of Humbert et al. (Humbert et al., 2002), theanti-phospho-huntingtin-S1181 and the anti-phospho-huntingtin-S1201described in the article of Anne et al. (Anne et al 2007). In aparticular embodiment, the cellular level of phosphorylated htt isobtained by measuring the staining intensity of cells in the sample.

In an embodiment, the method further comprises the step of comparing thecellular level of phosphorylated htt to a reference cellular level.Preferably, the reference cellular level is the cellular level ofphosphorylated htt in a normal sample. More preferably, the referencecellular level is the mean value of the cellular levels ofphosphorylated htt in a panel of normal samples. The normal sample is,as described above, a non-tumoral sample, preferably from the sametissue than the sample to be tested. The normal sample may be obtainedfrom the subject to be diagnosed or from another subject, preferably ahealthy subject.

In a further embodiment, the method further comprises the step ofdetermining whether the cellular level of phosphorylated htt is lowcompared to the reference cellular level. In a particular embodiment,the cellular level of phosphorylated htt in the sample to be tested isconsidered as low if the level is at least 2, 3, 4, 5, 6, 7, 8, 9 or10-fold lower than the reference cellular level.

In another embodiment, the present method further comprises assessing atleast one other marker used to diagnose cancer such as tumor grade,mitotic index, tumor size or expression of proliferation markers such asKi67, MCM2, CEA, CA19-9, CA125, PSA, β-hCG or CA15-3.

These markers are commonly used for diagnostic purposes and the resultsobtained with these markers may be combined with the results obtainedwith the present method in order to confirm the diagnosis.

The present invention also concerns a method for providing informationuseful for the diagnosis of a cancer in a subject, wherein the methodcomprises the step of determining the cellular level of phosphorylatedform of htt in a sample from said subject, a low cellular level ofphosphorylated htt indicating that said subject suffers from a cancer.

In an embodiment, the method further comprises the step of providing asample from the subject. Preferably, this sample is suspected to containtumoral cells.

In another embodiment, the method further comprises the step ofcomparing the cellular level of phosphorylated htt to a referencecellular level. Preferably, the reference cellular level is the cellularlevel of phosphorylated htt in a normal sample. More preferably, thereference cellular level is the mean value of the cellular levels ofphosphorylated htt in a panel of normal samples.

As demonstrated in the experimental section, the inventors show thatcells with decreased level of htt or decreased level of phosphorylatedhtt or with a poly-Q expansion of htt comprising more than 20 glutamineresidues exhibit increased cell motility and increased capacity ofmetastasis and invasion of surrounding tissues. The expression level ofhtt, the cellular level of phosphorylated htt and the number ofglutamine residues on the poly-Q expansion of htt thus constitute newmarkers for prognosis.

Accordingly, the present invention further concerns a method forpredicting, prognosing or monitoring clinical outcome of a subjectaffected with a cancer, wherein the method comprises the step ofdetermining the expression level of huntingtin in a cancer sample fromsaid subject, a low expression level of huntingtin being indicative of apoor prognosis.

In an embodiment, the method further comprises the step of providing acancer sample from the subject.

The expression level of htt can be determined from a cancer sample by avariety of techniques. In an embodiment, the expression level of htt isdetermined by measuring the quantity of htt protein or htt mRNA.

In a particular embodiment, the expression level of htt is determined bymeasuring the quantity of htt protein. The quantity of htt protein maybe measured by any methods known by the skilled person. Usually, thesemethods comprise contacting the sample with a binding partner capable ofselectively interacting with the htt protein present in the sample. Thebinding partner is generally a polyclonal or monoclonal antibody,preferably monoclonal. Polyclonal and monoclonal antibodies anti-htt arecommercially available such as anti-htt HU-4C8 (Euromedex), HU-2E8(Eurogentec), HU-2C1 (Euromedex), HU-4E6 (Millipore).

The quantity of htt protein may be measured by semi-quantitative Westernblots, enzyme-labeled and mediated immunoassays, such as ELISAs,biotin/avidin type assays, radioimmunoassay, immunoelectrophoresis orimmunoprecipitation. The protein expression level may be assessed byimmunohistochemistry on a tissue section of the cancer sample.Preferably, the quantity of htt protein is measured byimmunohistochemistry or semi-quantitative western-blot.

In another embodiment, the expression level of htt is determined bymeasuring the quantity of htt mRNA. Methods for determining the quantityof mRNA are well known in the art. For example the nucleic acidcontained in the sample (e.g., cell or tissue prepared from the patient)is first extracted according to standard methods, for example usinglytic enzymes or chemical solutions or extracted by nucleic-acid-bindingresins following the manufacturer's instructions. The extracted mRNA isthen detected by hybridization (e.g., Northern blot analysis) and/oramplification (e.g., RT-PCR). Preferably quantitative orsemi-quantitative RT-PCR is preferred. Real-time quantitative orsemi-quantitative RT-PCR is particularly advantageous. Preferably,primer pairs were designed in order to overlap an intron, so as todistinguish cDNA amplification from putative genomic contamination.Suitable primers may be easily designed by the skilled person. Othermethods of Amplification include ligase chain reaction (LCR),transcription-mediated amplification (TMA), strand displacementamplification (SDA) and nucleic acid sequence based amplification(NASBA). Preferably, the quantity of htt mRNA is measured byquantitative or semi-quantitative RT-PCR or by real-time quantitative orsemi-quantitative RT-PCR.

In an embodiment, the method further comprises the step of comparing theexpression level of htt to a reference expression level.

In a particular embodiment, the reference expression level is theexpression level of htt in a cancer sample without invasive ormetastatic capacity. Preferably, the reference expression level is themean value of expression levels of phosphorylated htt in a panel ofcancer samples without invasive or metastatic capacity. This sample maybe obtained from the subject to be diagnosed or from another subject.Preferably, this sample is from the same tissue than the sample to betested.

In a further embodiment, the method further comprises the step ofdetermining whether the expression level of htt is low compared to thereference expression level. In a particular embodiment, the expressionlevel of htt in the sample is considered as low if, after normalization,the level is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold lower than thereference expression level. Normalization of expression levels may beeasily done by any method known by the skilled person.

In another embodiment, the present method further comprises assessing atleast one other prognosis marker such as tumor grade, hormone receptorstatus, mitotic index, tumor size or expression of proliferation markerssuch as Ki67, MCM2, CEA, CA19-9, CA125, PSA, β-hCG or CA 15-3. Thesemarkers are commonly used and the results obtained with these markersmay be combined with the results obtained with the present method inorder to confirm the prognosis. The use of these markers is well-knownby the skilled person.

The present invention also concerns a method for predicting, prognosingor monitoring clinical outcome of a subject affected with a cancer,wherein the method comprises the step of determining the cellular levelof phosphorylated huntingtin in a cancer sample from said subject, a lowcellular level of phosphorylated huntingtin being indicative of a poorprognosis.

In an embodiment, the method further comprises the step of providing acancer sample from the subject.

The cellular level of phosphorylated form of htt may be determined byany method known by the skilled person. For instance, the cellular levelof phosphorylated form of htt may be determined by immunohistochemicalstaining such as described above.

In an embodiment, the method further comprises the step of comparing thecellular level of phosphorylated htt to a reference cellular level.

In a particular embodiment, the reference cellular level is the cellularlevel of phosphorylated htt in a cancer sample without invasive ormetastatic capacity. Preferably, the reference cellular level is themean value of cellular levels of phosphorylated htt in a panel of cancersamples without invasive or metastatic capacity. This sample may beobtained from the subject to be diagnosed or from another subject.Preferably, this sample is from the same tissue than the sample to betested.

In a further embodiment, the method further comprises the step ofdetermining whether the cellular level of phosphorylated htt is lowcompared to the reference cellular level. In a particular embodiment,the cellular level of phosphorylated htt in the sample to be tested isconsidered as low if the level is at least 2, 3, 4, 5, 6, 7, 8, 9 or10-fold lower than the reference cellular level.

In another embodiment, the present method further comprises assessing atleast one other prognosis marker such as tumor grade, hormone receptorstatus, mitotic index, tumor size or expression of proliferation markerssuch as Ki67, MCM2, CEA, CA19-9, CA125, PSA, β-hCG or CA15-3.

In another aspect, the present invention concerns a method forpredicting, prognosing or monitoring clinical outcome of a subjectaffected with a cancer, wherein the method comprises the step ofdetermining the number of glutamine residues on the poly-Q expansion ofhuntingtin in a sample from said subject, a poly-Q expansion comprisingmore than 20 glutamine residues being indicative of a poor prognosis.

In an embodiment, a poly-Q expansion comprising more than 35 glutamineresidues is indicative of a poor prognosis.

In another embodiment, a poly-Q expansion comprising more than 40glutamine residues is indicative of a poor prognosis.

The number of glutamine residues on the poly-Q expansion of huntingtinmay be determined by any method known by the skilled person. Forinstance, the htt encoding gene or a fragment thereof comprising thefirst exon may be amplified and sequenced to determine the number ofencoded glutamine residues.

The inventors demonstrate herein that polyQ expansion in htt comprisingmore than 35 glutamine residues is indicative of an increased likelihoodto have an earlier onset of cancer. Accordingly, the present inventionfurther concerns a method for determining the likelihood to have anearlier onset of cancer in a subject, wherein the method comprises thestep of determining the number of glutamine residues on the poly-Qexpansion of huntingtin in a sample from said subject, a poly-Qexpansion comprising more than 35 glutamine residues being indicative ofan increase likelihood to have an earlier onset of cancer. Inparticular, a poly-Q expansion comprising more than 35 glutamineresidues is indicative of an increased likelihood to have a cancerbefore 50 years old. It could be recommended to these subjects toperform earlier cancer screening tests than other subjects with a polyQexpansion in htt comprising 35 or less glutamine residues.

The present invention concerns a method for predicting or prognosing theaggressivity of a cancer in a subject affected with a cancer, whereinthe method comprises the step of determining the expression level ofhuntingtin in a cancer sample from said subject, a low expression levelof huntingtin being indicative of an aggressive cancer.

The present invention also concerns a method for predicting orprognosing the aggressivity of a cancer in a subject affected with acancer, wherein the method comprises the step of determining thecellular level of phosphorylated huntingtin in a cancer sample from saidsubject, a low cellular level of phosphorylated huntingtin beingindicative of an aggressive cancer.

The present invention further concerns a method for predicting orprognosing the aggressivity of a cancer in a subject affected with acancer, wherein the method comprises the step of determining the numberof glutamine residues on the poly-Q expansion of huntingtin in a samplefrom said subject, a poly-Q expansion comprising more than 20 glutamineresidues being indicative of an aggressive cancer. Preferably, a poly-Qexpansion comprising more than 35 glutamine residues is indicative of anaggressive cancer. More preferably, a poly-Q expansion comprising morethan 40 glutamine residues is indicative of an aggressive cancer.

In a further aspect, the present invention concerns a method forselecting a subject affected with a cancer for an antitumoral therapy,preferably an adjuvant chemotherapy and/or radiotherapy, or determiningwhether a subject affected with a cancer is susceptible to benefit froman antitumoral therapy, preferably an adjuvant chemotherapy and/orradiotherapy, wherein the method comprises the step of predictingclinical outcome of said subject by any one of methods of the inventionas described above, an indication of a poor prognosis indicating that anantitumoral therapy, preferably an adjuvant chemotherapy and/orradiotherapy, is required.

In an embodiment, the method for selecting a subject affected with acancer for an antitumoral therapy, preferably an adjuvant chemotherapyand/or radiotherapy, or determining whether a subject affected with acancer is susceptible to benefit from an antitumoral therapy, preferablyan adjuvant chemotherapy and/or radiotherapy, comprises the step ofdetermining the expression level of huntingtin in a cancer sample fromsaid subject as described above, a low expression level of huntingtinindicating that an antitumoral therapy, preferably an adjuvantchemotherapy and/or radiotherapy, is required.

In another embodiment, the method for selecting a subject affected witha cancer for an antitumoral therapy, preferably an adjuvant chemotherapyand/or radiotherapy, or determining whether a subject affected with acancer is susceptible to benefit from an antitumoral therapy, preferablyan adjuvant chemotherapy and/or radiotherapy, comprises the step ofdetermining the cellular level of phosphorylated huntingtin in a cancersample from said subject as described above, a low cellular level ofphosphorylated huntingtin indicating that an antitumoral therapy,preferably an adjuvant chemotherapy and/or radiotherapy, is required.

In a further embodiment, the method for selecting a subject affectedwith a cancer for an antitumoral therapy, preferably an adjuvantchemotherapy and/or radiotherapy, or determining whether a subjectaffected with a cancer is susceptible to benefit from an antitumoraltherapy, preferably an adjuvant chemotherapy and/or radiotherapy,comprises the step of determining the number of glutamine residues onthe poly-Q expansion of huntingtin in a sample from said subject asdescribed above, a poly-Q expansion comprising more than 20 glutamineresidues indicating that an antitumoral therapy, preferably an adjuvantchemotherapy and/or radiotherapy, is required. Preferably, a poly-Qexpansion comprising more than 35 glutamine residues indicates that anantitumoral therapy, preferably an adjuvant chemotherapy and/orradiotherapy, is required. More preferably, a poly-Q expansioncomprising more than 40 glutamine residues indicates that an antitumoraltherapy, preferably an adjuvant chemotherapy and/or radiotherapy, isrequired.

In a particular embodiment, the method further comprises assessing atleast one other cancer and prognosis marker such as tumor grade, hormonereceptor status, mitotic index, tumor size or expression ofproliferation markers such as Ki67, MCM2, CEA, CA19-9, CA125, PSA,(β-hCG or CA15-3. The results obtained with these markers may be used toconfirm the result obtained with the method according to the inventionand/or to orientate the choice of the adjuvant therapy.

The present invention also concerns a method for selecting, identifyingor screening a compound useful for treating a subject having cancer,wherein the method comprises the selection or identification of acompound capable of increasing the expression level and/or thephosphorylation of huntingtin.

In an embodiment, the method comprises:

a) providing a huntingtin protein or a fragment thereof of at least 50consecutive amino acids and comprising at least one phosphorylatedresidue selected from the group consisting of S421, S535, S1181, S1201,S2076, S2653 and S2657;

b) providing a compound dephosphorylating at least one phosphorylatedresidue comprised in htt protein or the fragment thereof provided instep a);

c) contacting a candidate compound with said huntingtin protein orfragment thereof and said dephosphorylating compound; and,

d) selecting the candidate compound that inhibits the dephosphorylationof at least one phosphorylated residue comprised in htt protein or thefragment thereof provided in step a) by dephosphorylating compound.

In a particular embodiment, the method comprises:

a) providing a huntingtin protein or a fragment thereof of at least 50consecutive amino acids and comprising at least a phosphorylated residueat position S421;

b) providing a calcineurin;

c) contacting a candidate compound with said huntingtin protein orfragment thereof and said calcineurin; and,

d) selecting the candidate compound that inhibits the dephosphorylationof the phosphorylated residue of huntingtin at position S421 bycalcineurin.

In another embodiment, the method comprises:

a) contacting a candidate compound with a cell expressing a huntingtinprotein and comprising a kinase which phosphorylates huntingtin at aposition selected from the group consisting of S421, S535, S1181, S1201,S2076, S2653 and S2657, and a compound dephosphorylating thephosphorylated residue at selected position;

b) assessing the amount of huntingtin phosphorylated and/or the amountof huntingtin which is not phosphorylated; and,

c) selecting the candidate compound that increases the phosphorylationof huntingtin at selected position in comparison with a control cellwhich has not been contacted with the candidate compound.

In a further embodiment, the method comprises:

a) contacting a candidate compound with a cell expressing a huntingtinprotein and comprising a kinase which phosphorylates huntingtin atposition S421 and a calcineurin;

b) assessing the amount of huntingtin phosphorylated and/or the amountof huntingtin which is not phosphorylated; and,

c) selecting the candidate compound that increases the phosphorylationof huntingtin at position S421 in comparison with a control cell whichhas not been contacted with the candidate compound.

The phosphorylation of htt may be detected with an anti-phosphorylatedhtt antibody such as described above.

In a further embodiment, the method comprises:

a) contacting a candidate compound with a cell expressing a huntingtinprotein;

b) assessing the amount of huntingtin expressed in said cell; and

c) selecting the candidate compound that increases the expression ofhuntingtin in comparison with a control cell which has not beencontacted with the candidate compound.

In a last aspect, the present invention concerns a kit:

(a) for diagnosing or detecting a cancer in a subject; and/or

(b) for predicting, prognosing or monitoring clinical outcome of asubject affected with a cancer; and/or

(c) for selecting a subject affected with a cancer for an adjuvanttherapy and/or radiotherapy or determining whether a subject affectedwith a cancer is susceptible to benefit from an adjuvant chemotherapyand/or radiotherapy; and/or

wherein the kit comprises:

(i) at least one antibody specific to htt and, optionally, means fordetecting the formation of the complex between htt and said at least oneantibody; and/or

(ii) at least one antibody specific to phosphorylated htt and,optionally, means for detecting the formation of the complex between httand said at least one antibody; and/or

(iii) at least one probe specific to the protein htt and, optionally,means for detecting the hybridization of said at least one probe on httprotein; and/or

(iv) at least one nucleic acid primer pair specific to htt gene or mRNAand, optionally, means for amplifying and/or detecting said gene or saidmRNA; and,

(v) optionally, a leaflet providing guidelines to use such a kit.

All references cited in this specification are incorporated byreference.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.”

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgement or admission or any formof suggestion that that prior publication (or information derived fromit) or known matter forms part of the common general knowledge in thefield of endeavour to which this specification relates.

Further aspects and advantages of the present invention will bedescribed in the following examples, which should be regarded asillustrative and not limiting.

EXAMPLES Example 1 HTT and Breast Cancer

Materials and Methods

Antibodies—The antibodies used herein are as follows: monoclonalanti-Htt 4c8 (Euromedex); rabbit anti-Htt 737 (Anne et al; 2007); rabbitanti-phospho-S421-human Htt 763 (Humbert et al, 2002); monoclonalβ-actin (Sigma); monoclonal ZO-1 (BD Transduction Laboratories); rabbitanti-firefly luciferase (Abeam); and monoclonal anti-GM-130 (BDTransduction Laboratories). Secondary antibodies for immunofluorescencestudies were Alexa 488 (anti-mouse and anti-rabbit), 555 (anti-mouse andanti-rabbit), and Cy5 anti-mouse from Invitrogen. Secondary antibodiesfor western blots were HRP-conjugated anti-mouse and anti-rabbit fromInvitrogen.

Cells—4t1-luc and 67NR cells were maintained in DMEM (Gibco) with 10%fetal calf serum, 1×MEM non-essential amino acids (Gibco), 100 units/mLpenicillin/streptomycin (Gibco), 250 ng/mL Fungizone (Gibco), and 400μg/ml Hygromycin B (Invitrogen). MCF7, CAMA-1 and HEK293 cells weremaintained in DMEM with 10% fetal calf serum, 100 units/mLpenicillin/streptomycin (Gibco), and 0.1% Fungizone (Gibco). COS-7 cellswere maintained in DMEM with 10% fetal calf serum, 100 units/mLpenicillin/streptomycin (Gibco), 1% glutamine, and 250 ng/mL Fungizone(Gibco). 2A1 cells were maintained in DMEM with 10% bovine calf serum,100 units/mL penicillin/streptomycin (Gibco), 1% glutamine, 250 ng/mLFungizone (Gibco), and 400 μg/ml geneticin (Gibco). SH-SY5Y cells weremaintained in RPMI with 10% BCS and 1% glutamine.

2A1 cells were grown at 33° C. and 5% CO₂. All other cells were kept at37° C. and 5% CO₂.

Plasmids, siRNA and transfections—The hairpin region of shHtt recognizesa region within exons 8-9 (AGCTTTGATGGATTCTAATCTCTTGAAATTAGAATCCATCAAAGCT, SEQ ID NO: 4). Within the same plasmid is a separate open readingframe coding for GFP behind the PGK promoter. The shLuc construct isidentical apart from its hairpin recognizing a sequence within thefirefly luciferase gene rather than a sequence within htt. The 17QHtt480plasmids are described in Saudou et al., 1998. mCherry is expressed inthe pCDNA 3.2 plasmid. The pARIS-Htt plasmids used herein are variationsof a synthetic full-length htt gene with multiple tags in the pCDNA 3.2plasmid. This plasmid is rendered insensitive to multiple siRNA andshRNA sites, including those used herein, by point mutations in the DNAwhich do not affect the amino acid sequence. The S421A and S421Dmutations were introduced by simple site-directed mutagenesis using theInvitrogen Gateway System.

The siRNA against mouse Htt used herein, previously described in Zala etal., 2008, is complementary to the coding sequence 361-380 of mouse Htt.The control scRNA is a scrambled version of the nucleotides in the siRNAverified to not have complementarity to any other sites in the genome.

Transfections were carried out as follows. For random migration assaysin 2A1, cells were lipofected (LIPOFECTAMINE 2000, Invitrogen) orelectroporated 2-3 days before assays with either shHTT or shLuc andpCDNA3.2-cherry, or CTFLHtt-WT, -S421A, or -S421D. MCF-7 cells werelipofected 48 hours before scratch assays and before lysis forimmunoprecipitation experiments. COS-7 cells were electroporated 24-48hours before fixation for leading edge quantification studies. HEK293cells were transfected by calcium phosphate precipitation. siRNAtreatment of 4t1 cells was carried out using lipofectamine 2000.

Infection by lentiviral vectors—Using the Gateway subcloning system fromInvitrogen, CTFLhtt constructs were cloned into a lentiviral vector.This vector was then transfected into HEK 293T cells. Cells were lysed,and lentiviral particles were collected. Titer was calculated by ELISAassay. 4t1-luc cells in 24-well plates were then infected with 1.125 ugof virus. The media was changed 24 hours later.

Generation of stable cell lines—Expression of CTFLhtt, as observed byfluorescence microscopy, revealed a high but incomplete lentiviralinfection rate of 4t1-luc cells, so fluorescent activated cell sortingwas carried out to purify the cell population. Subsequentimmunocytochemical and immunoblotting studies suggested that the rate ofCTFLhtt expressing cells was close to 100% and that the levels ofexpression between the different cell lines were similar.

Immunohistochemistry—Paraffin imbedded biopsies from breast cancerpatients and healthy subjects were obtained from the Curie InstituteHospital pathology department. All samples were de-paraffinized and thende-masked in Dako EDTA pH 9 antigen retrieval solution at 90° C. for 30minutes. Samples were then probed using a Vectastain ABC kit from Vectorlaboratories. The 4c8 anti-Htt antibody was used at a concentration of1:300, and the phospho-Htt 763 antibody was used at a concentration of1:50. All samples were also counterstained with Hematoxylin to identifynuclei.

Immunofluorescence—To examine the total level of htt in 2a1 cells whichhad been gene-replaced, cells were fixed with cold methanol and thenstained with 737 total htt antibody (1:25). Gene-replaced cells wereidentifiable by the GFP fluorescence from the shHtt and the cherryfluorescence from the CTFLhtt.

To verify whether 4t1-luc cells from the in vivo metastasis assay wereindeed knocked down for htt from the intraperitoneal injections ofsiHtt, lungs were dissected and then frozen in liquid nitrogen. Sampleswere then processed for immunofluorescence imaging by cryostatsectioning and adhering to Thermo Scientific Superfrost glass slides.Sections were then fixed in 4% paraformaldehyde and blocked for threedays to remove any autofluorescence in 1% BSA+0.1% triton x-100+0.15%glycine. Samples were then probed with antibodies against luciferase(1:500) and htt (4c8 1:400). Imaging was then carried out using a LeicaTCS-SP5 confocal microscope with 63× objective lens.

Cell Motility—Random migration experiments were carried out in 2a1 andMCF7 cells. Cells in plastic 6-well plates were transfected, asdescribed above, and then imaged over at least six hours using aninverted fluorescent 2D Leica DM IRB microscope with photometricCoolSNAP fx camera in a chamber with controlled temperature and CO₂conditions and a moving stage. This system allows for the storage ofcell coordinates so that many cells at different positions can befollowed during the same time-course. Transfected cells were identifiedby fluorescence imaging as described above. Dividing cells were excludedfrom analysis. shLuc- or shHtt-transfected MCF7 cells were induced tomove as follows. Cells were trypsinized 16 hours before assay and seededonto wells of a fibronectin-coated (20 μg/mL) plastic 6-well plate. EGF(Sigma) was then added to 20 ng/mL. Imaging began one hour later usingthe same protocol as for the 2a1 cells. The cell tracking plug-in ofImageJ software was then used to calculate parameters of cell motility.

Scratch assays were performed in 4t1 and MCF7 cells. Transfected cellswere grown 2-3 days until confluence. Then, a 200 μL pipet tip was usedto scratch the confluent monolayer. Cells were then imaged for 48 hoursusing the same microscope configuration as described for randommigration assays. ImageJ software was then used to measure the distancebetween the two columns of cells at specific timepoints. In all cases,the in vitro doubling times for the cells within each condition wereconfirmed to be the same so treatment with inhibitors of cell divisionwere not necessary.

Matrigel Invasion—Growth factor reduced Matrigel invasion chambers werefrom BD Biosciences (Reference number 354483). Before addition of cells,chambers were allowed to warm to room temperature for one hour and thenre-hydrated in 0.1% serum containing 40 media in a 37° C. incubator at5% CO₂ for at least three hours. Just before the addition of cells, thechambers were placed into wells containing full serum (10%) containing411 media. 4t1-luc cells were treated with siRNA or scramble for 24hours before being added to the chambers. Cells were serum starved in0.1% serum-containing 4t1 media for at least six hours before beingtrypsinized and resuspended. 100,000 cells were then resuspended in 500μL 0.1% serum 4t1 media and added to the top of the chambers. 24 hourslater, the matrigel was removed from the chamber, the chamber was rinsedtwice with 0.1% serum 4t1 media, and the chambers were fixed in 4%paraformaldehyde for 10 minutes. The chambers were then washed threetimes in 1×PBS. Next, the chambers were stained with toluidene blue dyefor 30 minutes and washed 5 times with water over the course of tenminutes. Then, images were taken of the chambers, still housed in24-well dishes, using an inverted microscope. The cells were thencounted using an ImageJ cell counter plug-in.

In vivo metastasis—The in vivo metastasis assay was done using 4t1-luccells injected into syngenic BALB/C mice as described previously(Fitamant et al., 2008) Briefly, 100,000 cells resuspended in 150 μL ofPBS were tail-vein injected. 4 μg of sc or siRNA was intraperitoneallyinjected each day for ten days, starting with the day of the cellinjections. Each day, beginning at day 4, luciferin was administered tothe mice, and bio-luminescence imaging was performed, and the lungsignal was quantified. Statistical differences in lung signal intensitywere determined by Mann-Whitney U post-hoc analysis. The siRNA used inthis study was the mouse siHtt described previously. The scramblecontrol was a previously used sequence containing no homology to knownsites of the mouse genome (GAUAGCAAUGACGAAUGCGUA, SEQ ID NO: 5).Survival Assays were performed in compliance with French animal researchethical standards: when mice lost more than 15% of their pre-treatmentweight due to the effects of the cancer cells, they were euthanized. Theday of euthanasia was recorded as their last day of survival. Controlmice treated with sc or siRNA, but never injected with 4t1-luc cells,never showed any loss of weight during the course of the study.Kaplan-Meier survival plots were evaluated by a log-rank analysis forstatistical significance.

Results

Htt is Present at High Levels in Normal Breast Epithelia and CancerCells

While the expression of Htt is known to be ubiquitous, it is thought tobe enriched in the brain. However, the inventors found that htt ispresent in breast cells lines at a similar level as in neuronal celllines (FIG. 1A). The finding of a high endogenous level of htt incultured breast cancer cells prompted an examination of the localizationof Htt protein in normal breast tissue. It was discovered that htt wasexpressed specifically in the epithelial cells lining both mammaryglands and ducts and not detectable in stromal cells (FIG. 1C).

The state of htt and its phosphorylation at serine 421 (S421), amodification known to regulate htt toxicity and function, were examinedin human breast cancer. To this end, the inventors tested viaimmunohistochemistry and western blot analysis whether htt was enrichedin breast tumor samples. Western blotting revealed that htt was greatlyenriched in samples from breast tumor samples as compared to healthybreast tissue from the same individuals (FIG. 1B). However, one can notconclude from these studies whether the enrichment of htt in tumorsamples reflects a cellular upregulation of htt protein in the cancerstate. In fact, based on immunohistochemical studies, it seems morelikely that the enrichment is simply a result of the tumor itself beinga proliferation of cells of epithelial origin (compare FIG. 1C to FIG.1E).

In order to examine the differences in the phosphorylation of S421 inthe normal versus cancer conditions, the inventors performed stainingswith an antibody (763) specific to the phosphorylated form of htt (pHtt)in normal (FIG. 1D) and cancerous breast tissue (FIGS. 1F and G). Theysurprisingly found a very specific staining of pHtt to small punctatestructures at the apical part of the junctions between breast epithelialcells (FIG. 1D, inset). It was hypothesized that these structures couldpossibly be adherens junctions or tight junctions.

To investigate whether total htt or pHtt were differentially regulatedin the cancer state, immunohistochemical staining of normal tissue wascompared to that of cancerous tissue. No difference was found betweenthe intensity of 4C8 staining between normal cells and cancerous ones(FIGS. 1C and E), but a striking difference was observed in pHttstaining when comparing normal to cancerous cells (FIGS. 1D,E,F). Theoverall trend the inventors noticed was that htt phosphorylation waslost in invasive cancer cells and, to a lesser degree, in primary tumors(FIGS. 1H and F, respectively). The loss of phosphorylation wasquantified in invasive cancer cells in breast cancer patient samplesthat contained both invasive cancer cells and normal glands or ducts asan internal positive control for staining. It was found that, thoughnormal htt staining was present in the invasive cells of all breastcancer samples examined, pHtt staining was lost in 6/9 cases of ductalbreast cancer (FIG. 1I) and 10/10 cases of lobular disease (FIG. 1J). Amore general correlation was also noticed between the level ofdifferentiation of breast epithelial cells and the presence of httphosphorylation at residue S421 (data not shown).

Htt Regulates Cell Motility

The processes of in vivo invasion and in vitro cell motility share manyof the same signaling pathways. In vitro assays were designed todetermine whether htt played any role in cell motility. The firstapproach was a simple scratch assay in MCF7 breast cancer cells. Cellswere transfected with either a control small hairpin RNA (shRNA)construct against firefly luciferase (shLuc) or an shRNA against htt(shHtt) to reduce endogenous levels of the protein. Both of theseplasmids contain a gene for green fluorescent protein (GFP) behind aseparate promoter, allowing the identification of live transfected cells(FIG. 2A, middle column). After transfection, cells were grown toconfluence and then scratched with a pipette tip. Closure of the scratchwas monitored by live cell imaging and quantified using ImageJ software.It was found that cells in which the endogenous htt was decreased byshHtt showed a faster scratch closure than control cells, suggestingthat htt does play a role in cell motility (FIG. 2B). A similar scratchclosure assay in 4t1 mouse breast cancer cells transfected by a scrambleRNA or an siRNA against htt supported this conclusion (FIG. 2C).

While scratch closure assays are well established to evaluate cellmotility in epithelial cells, they likely under-estimate the totaleffect when cells are transiently transfected with sh- or siRNA due toincomplete transfection. Another limitation is that scratch closurereflects collective cell movement, as opposed to individual cellmovement. To address these problems, random cell migration assays wereperformed in MCF7 cells. To induce individual MCF7 cells to move,plastic culture plates were coated with fibronectin, cells were platedat low density and later epidermal growth factor (EGF) was added. Cellswere again transfected with either shLuc or shHtt. Western blot analysis(not shown) and immunofluorescence studies (FIG. 2A, right column)validated the efficacy of shHtt to reduce endogenous htt levels. Theresults of this assay further supported the previous motilityexperiments in that cells expressing shHtt moved about 50% faster thanthe shLuc-expressing control cells.

Though these results were consistent with a role for htt in regulatingcell motility, the analysis of the random MCF7 cell migration assay maysuffer potential confounds due to the stimulation of cells byfibronectin and EGF. Therefore, a similar experiment was carried out innon-epithelial cells which exhibit individual cell movement withoutneeding to be stimulated by any growth factors. 2a1 cells, a neuronalcell line well established for the study of the htt protein, werechosen. This assay simply required that cells be transfected with shLucor shHtt and then followed by live cell imaging. As with the MCF7 cells,2a1 cells expressing shHtt moved about 50% faster than the controlshLuc-expressing cells (FIG. 2E, bars shLuc and shHtt)).

Taken together these four separate motility assays demonstrate thatdecreasing endogenous htt leads cells to move faster.

The inventors' hypothesis based on the in vivo invasion data (FIG. 1)was that when cells in culture lose phosphorylated htt, they would movefaster. In order to test this hypothesis, a “gene replacement”experiment was designed. Co-transfections were carried on with shHtt andone of three cherry-tagged, shRNA-resistant full-length Htt plasmids(CTFLhtt): wildtype (WT), a point mutant at serine 421 to an alanine tomimic constitutive dephosphorylation (S421A) or a point mutant at serine421 to an aspartic acid to mimic constitutive phosphorylation (S421D).In this way, the cellular pool of full-length htt could be manipulatedto reflect conditions of high or low phosphorylation of htt at S421. Asthese cells would express both GFP to identify endogenous-htt depletedcells and cherry to identify cells expressing the point mutants, it waspossible to verify that the “gene-replaced” cells expressed levels oftotal htt similar to untransfected cells (FIG. 1A, lower right panel).

It was assumed that, if phosphorylation of Htt is required formaintaining a basal level of cell motility, then the expression of theWT or S421D constructs would rescue the increased cell motility broughtabout by decreasing endogenous htt, while the S421A construct would not.The results of the experiment closely agreed with this idea (FIG. 2E).This experiment also rules out the possibility that the increasedmotility seen in shHtt-expressing cells could be due to off-targeteffects of the shRNA construct (FIG. 2E, bar shHtt vs. bar shHtt+WT).

In conclusion, some level of htt phosphorylation is required to maintainnormal cell motility in vitro. When this level is reduced, for exampleby shHtt, cells move faster.

Htt Regulates Metastasis In Vivo

Based on the finding in human breast cancer patients that httphosphorylation is lost in invasive cancer cells and on the finding thatthe increased cell motility which results from decreasing endogenous httcan be rescued only by phosphorylated htt, an in vivo experiment wasdesigned to examine whether htt could also regulate metastasis. The4t1-luciferase system was chosen to test this possibility. In this wellestablished experimental paradigm, syngenic mice are tail-vein injectedwith stably luciferase-expressing breast cancer cells (4t1-luc), andmetastasis to lung is monitored by live bioluminescence imagingfollowing systemic injection of luciferin (Fitamant et al., 2008).

To determine whether htt could modulate metastasis in this system, micewere injected with a scramble control (sc) or an siRNA against htt(siHtt) (FIG. 3A). Seven days after the cancer cell injections, astriking ten-fold increase was found in the luciferase signal in thelungs of siHtt-treated mice as compared to scramble-treated mice (FIGS.3B,C). Immunofluorescence evaluation of the lungs of siHtt-treated micerevealed that the luciferase-positive 4t1 cells did indeed show asubstantially decreased level of htt as compared to control mice (FIG.3E). Gross evaluation of these dissected lungs indicated a substantiallyhigher level of metastasis in the siHtt-treated mouse. Furthermore,these data correlate well with a statistically significant decrease inthe survival of the siHtt-treated mice (FIG. 3D). No siHtt-treated micenot injected with 4t1-luc cells died during the course of theexperiment, confirming that this decreased survival was due to an effectof the siHtt on cancer progression in these mice.

Htt Phosphorylation Controls Cancer Cell Invasion

To definitively understand whether changes in htt were causative eventsin cancer cell invasion or simply silent downstream targets of the trueeffectors, the inventors tested how manipulating htt might alter cancercell invasion in matrigel invasion chambers. As in the above motilityexperiments, the influence of knocking down endogenous htt and replacingit with the different CTFLhtt constructs mutated at S421, wasspecifically examined. To this end, 4t1-luc cell lines stably expressingthe three different CTFLhtt constructs: WT, S421A and S421D, werecreated. These constructs are resistant to siRNA. Thus, a “genereplacement” strategy was used to examine the effects of httphosphorylation on invasion.

The results of this experiment demonstrate that when a cell has a higherproportion of unphosphorylated Htt, it is much more capable of invasion.Specifically, 4t1-luc cells which have been gene-replaced with theCTFLS421A mutant show a >15 fold increase in the number of cells whichwere able to invade through matrigel chambers compared to non-genereplaced 4±1-luc cells (GFP scramble) (FIG. 3F). No such effect was seenin cells which had been gene-replaced for the S421D mutant. Thisdemonstrates that cells require some level of phosphorylated htt toprevent elevated invasive ability. When this relative level isdecreased, cells become much more invasive.

Example 2 PolyQ-Htt and Breast Cancer

Materials and Methods

Mice—Mice were housed in a 12 hour light/dark cycle and fed a regulardiet and water ad libitum. Mice were sacrificed by cervical dislocation.Mammary glands were dissected and spread on glass slides. Mammary glandswere fixed overnight in methacarn (60% methanol, 30% chloroform and 10%acetic acid), washed in 100% methanol for 1 hour and left overnight infresh methanol. For whole mount staining, mammary glands were rehydratedby a bath of 70% ethanol for 15 min, followed by a 5 min bath in H2O andstained overnight with carmine aluminum staining. Stained glands weredehydrated by 15 min baths in ethanol (70%, 95% and 100%), cleared inxylene, and stored in methyl salicylate. Else, paraffin embedded tissuewas fixed and stained with hematoxylin and eosin (Taddei et al., 2008).Grafts experiments were performed as previously described for xenografts(Morton and Houghton, 2007) except that 8 mm³ pieces of mouse tumorswere grafted in 6 week-old Swiss nude mice.

Random migration assays—Cells in plastic 6-well plates were imaged over6-8 hours using an inverted fluorescent 2D Leica DM IRB microscope withphotometric CoolSNAP fx camera in a chamber with controlled temperatureand CO₂ conditions and a moving stage. This system allows for thestorage of cell coordinates so that many cells at different positionscan be followed during the same time-course. Transfected cells wereidentified by fluorescence imaging as described above. Dividing cellswere excluded from analysis. The cell tracking plug-in of ImageJsoftware was then used to calculate parameters of cell motility.

Invasion assays—Growth factor reduced Matrigel invasion chambers werefrom BD Biosciences (Reference number 354483). Before addition of cells,chambers were allowed to warm to room temperature for one hour and thenre-hydrated in 0.1% serum containing culture media in a 37° C. incubatorat 5% CO₂ for at least three hours. Just before the addition of cells,the chambers were placed into wells containing 10% serum and culturemedia. Cells were serum starved in 0.1% serum-containing media for atleast six hours before being trypsinized and resuspended. 100,000 cellswere then resuspended in 500 μl of media containing 0.1% serum and addedto the top of the chambers. 24 hours later, the matrigel was removedfrom the chamber, the chamber was rinsed twice with 0.1% serum media,and the chambers were fixed in 4% paraformaldehyde for 10 minutes. Thechambers were then washed three times in 1×PBS, stained with toluideneblue dye for 30 minutes and washed 5 times with water over the course often minutes. Then, images were taken of the chambers, still housed in24-well dishes, using an inverted microscope. The cells were thencounted using an ImageJ cell counter plug-in.

Results

Oncogene-Induced Mammary Tumors Develop Faster in HD Mouse Model

To examine the influence of the presence of polyQ-huntingtin on tumorprogression, the inventors derived mice that express activated oncogenesand polyQ-huntingtin. Mice expressing the activated PyVT (Guy et al.,1992) or ErbB2 (Muller et al., 1988) under the control of the MMTVpromoter (respectively MMTV-PyVT and MMTV-ErbB2) were crossed with themouse line carrying a 111 CAG expansion inserted into the endogenousmouse huntingtin gene (Wheeler et al., 2002). Compared with MMTV-PyVTmice expressing wild-type huntingtin (MMTV-PyVT; Hdh^(Q7/Q7)), theappearance of tumors was observed earlier in MMTV-PyVT; Hdh^(Q111/Q111)mice (FIG. 4A). Similar results were obtained using the MMTV-ErbB2 modelof mammary cancer in mouse (FIG. 4A). Examination of virgin femalemammary whole mount from MMTV-PyVT; Hdh^(Q111/Q111) (FIG. 4B) andMMTV-ErbB2; Hdh^(Q111/Q111) (FIG. 4B) revealed the presence of multiplelarge mammary adenocarcinomas as compared to the adenocarcinomas inducedin the huntingtin wild-type background. These observations suggest thatthe presence of polyQ huntingtin accelerates mammary carcinogenesis inoncogene-induced mouse models.

PolyQ Tumors do not Grow Faster but Show Increased Activation of Akt

The inventors next evaluated the growth of tumors in wild-type and HDcontext by grafts experiments. Primary solid-tumor isolates fromMMTV-PyVT; Hdh^(Q7/Q7) and MMTV-PyVT; Hdh^(Q111/Q111) were transplantedin immunodeficient mice and tumor size was measured as a function oftime following transplantation. In both wild-type and HD background, theprimary tumors grew linearly at similar rate (FIG. 5) showing that thefaster appearance of tumors in HD models is not due to acceleratedgrowth of the tumors.

PolyQ-Huntingtin Increases Breast Cancer Cell Motility, Invasiveness andMetastasis

The inventors next derived primary tumors cells from MMTV-pyVT;Hdh^(7/Q7)/Q⁷ and MMTV-PyVT; Hdh^(Q111/Q111) tumors and performed randomcell migration assays (FIG. 6A). Cells expressing the polyQ form ofhuntingtin moved faster than the control cells. The inventors alsotested how polyQ huntingtin might alter cancer cell invasion in matrigelinvasion chambers (FIG. 6B). In this assay, the primary cells derivedfrom MMTV-PyVT; Hdh^(Q111/Q111) tumors show an increase in the number ofcells which were able to invade through matrigel chambers compared toprimary cells derived from MMTV-PyVT; Hdh^(Q7/Q7) tumors.

Based on the findings that polyQ-huntingtin leads to increasedinvasiveness, the inventors next examined metastasis to the lungs inimmunodeficient mice grafted with primary solid-tumor isolates fromMMTV-ErbB2; Hdh^(Q7/Q7) and MMTV-ErbB2; Hdh^(Q111/Q111) (FIG. 6C).Thirty-eight days after the grafts, lungs were dissected, sectioned andstained with hematoxylin and eosin. In animals grafted with MMTV-ErbB2;Hdh^(Q111/Q111) tumors, the metastasis was increased as compared toanimals grafted with MMTV-ErbB2; Hdh^(Q7/Q7) tumors.

Thus, cells expressing polyQ-huntingtin are more motile and invasivethan control tumor cells expressing wild-type huntingtin and this leadsto increased metastasis in vivo in the polyQ context.

HD Patients Develop More Aggressive Forms of Breast Cancer than Non HDPatients

To establish the physiological relevance of the inventors' findings,they identified nine HD patients with breast cancer. The examination oftheir medical records revealed that they developed more aggressive formsof cancer. Indeed, the majority of them were grade III with metastasis(data not shown). In HD, there is an inverse correlation between thelength of the CAG repeat and the age of onset with long expansionscorresponding to juvenile forms of the disease (Young, 2003). Here, theinventors observed a striking correlation between the age of canceronset and the polyglutamine length (FIG. 7). Together, theseobservations are in agreement with the mouse models data, showing thatthe abnormal polyQ expansion causing HD leads to more severe progressionof breast cancer.

In conclusion, these results demonstrate that polyQ expansion in httincreases tumorigenesis and metastasis capacity.

Example 3 HTT and Prostate Cancer

Materials and Methods

siRNA and transfections—A small hairpin RNA construct against htt(shHtt) was used. The hairpin region of shHtt recognizes a region withinexons 8-9 (AGCTTTGATGGATTCTAATCTCTTGAAATTAGAATCCATCAAAGCT, SEQ ID NO:4). The shLuc construct is identical apart from its hairpin recognizinga sequence within the firefly luciferase gene rather than a sequencewithin htt.

Transfections were performed as described in example 1.

Cell line—The LNCaP human prostate cell line was used. The LNCaP cellline was established from a metastatic lesion of human prostaticadenocarcinoma.

Cell motility—Random migration assays were performed as described inexample 1.

Results

shHtt has been used to decrease levels of htt in LNCaP human prostatecell line as described in example 1. The migration of these cells wasthen assessed (random migration assays) and compared to control cells(LNCap cells transfected with shLuc). It was observed that decreasinghtt level induces an increased capacity of cells to move.

This demonstrates that htt is involved in motility of prostate cancercells and that the dysregulation of this function participates to themetastasis and invasive capacities of these cells.

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1. A method for diagnosing or detecting a cancer in a subject, wherein the method comprises the step of determining the cellular level of phosphorylated form of huntingtin in a sample from said subject, a low cellular level of phosphorylated huntingtin indicating that said subject suffers from a cancer.
 2. The method according to claim 1, wherein the method further comprises the step of comparing the cellular level of phosphorylated huntingtin to a reference cellular level
 3. The method according to claim 2, wherein the reference cellular level is the cellular level of phosphorylated huntingtin in a normal sample.
 4. A method for predicting, prognosing or monitoring clinical outcome of a subject affected with a cancer, wherein the method comprises the step of determining (i) the expression level of huntingtin or (ii) the cellular level of phosphorylated huntingtin, in a cancer sample from said subject or (iii) the number of glutamine residues on the poly-Q expansion of huntingtin in a sample from said subject, a low expression level of huntingtin, a low cellular level of phosphorylated huntingtin or a poly-Q expansion comprising more than 20 glutamine residues, being indicative of a poor prognosis.
 5. The method according to claim 4, wherein the method further comprises the step of comparing the expression level of huntingtin or the cellular level of phosphorylated huntingtin to a reference expression level.
 6. The method according to claim 4, wherein a poly-Q expansion comprising more than 35 glutamine residues is indicative of a poor prognosis.
 7. The method according to claim 4, wherein a poor prognosis is a decreased patient survival and/or an early disease progression and/or an increased metastasis formation.
 8. A method for selecting a subject affected with a cancer for an antitumoral therapy or determining whether a subject affected with a cancer is susceptible to benefit from an antitumoral therapy, wherein the method comprises the step of determining (i) the cellular level of phosphorylated huntingtin or (ii) the expression level of huntingtin in a cancer sample from said subject or (iii) the number of glutamine residues on the poly-Q expansion of huntingtin in a sample from said subject, a low cellular level of phosphorylated huntingtin, a low expression level of huntingtin or a poly-Q expansion comprising more than 20 glutamine residues indicating that an antitumoral therapy is required.
 9. A method for selecting, identifying or screening a compound useful for treating a subject having cancer, comprising the selection or identification of a compound capable of increasing the expression level and/or the phosphorylation of huntingtin.
 10. The method according to claim 9, wherein said method comprises: a) providing a huntingtin protein or a fragment thereof of at least 50 consecutive amino acids and comprising at least one phosphorylated residue selected from the group consisting of S421, S535, S1181, S1201, S2076, S2653 and S2657; b) providing a compound dephosphorylating at least one phosphorylated residue comprised in htt protein or the fragment thereof provided in step a); c) contacting a candidate compound with said huntingtin protein or fragment thereof and said dephosphorylating compound; and d) selecting the candidate compound that inhibits the dephosphorylation of at least one phosphorylated residue comprised in htt protein or the fragment thereof provided in step a) by dephosphorylating compound.
 11. The method according to claim 9, wherein said method comprises: a) contacting a candidate compound with a cell expressing a huntingtin protein and comprising a kinase which phosphorylates huntingtin at a position selected from the group consisting of S421, S535, S1181, S1201, S2076, S2653 and S2657, and a compound dephosphorylating the phosphorylated residue at selected position; b) assessing the amount of huntingtin phosphorylated and/or the amount of huntingtin which is not phosphorylated; and c) selecting the candidate compound that increases the phosphorylation of huntingtin at selected position in comparison with a control cell which has not been contacted with the candidate compound.
 12. The method according to claim 9, wherein said method comprises: a) contacting a candidate compound with a cell expressing a huntingtin protein; b) assessing the amount of huntingtin expressed in said cell; and c) selecting the candidate compound that increases the expression of huntingtin in comparison with a control cell which has not been contacted with the candidate compound.
 13. A method for treating cancer in a subject, comprising administering a therapeutically effective amount of a compound increasing the cellular level of the phosphorylated form of huntingtin.
 14. The method according to claim 13, wherein the huntingtin protein is phosphorylated at one or several positions selected from the group consisting of S421, S535, S1181, S1201, S2076, S2653 and S2657.
 15. The method according to claim 14, wherein the huntingtin protein is phosphorylated at position S421.
 16. The method according to claim 13, wherein said compound is selected from the group consisting of huntingtin protein and a biologically active fragment thereof, huntingtin protein comprising the mutation S241D and a biologically active fragment thereof, and a nucleic acid encoding thereof.
 17. The method according to claim 13, wherein said compound inhibits the dephosphorylation of huntingtin.
 18. The method according to claim 17, wherein said compound is a calcineurin inhibitor or a compound inhibiting the interaction between calcineurin and huntingtin.
 19. The method according to claim 18, wherein the calcineurin inhibitor is a nucleic acid molecule interfering specifically with calcineurin expression, preferably a RNAi, an antisense nucleic acid or a ribozyme.
 20. The method according to claim 18, wherein the calcineurin inhibitor is a dominant-interfering form of calcineurin.
 21. The method according to claim 13, wherein said compound increases the phosphorylation of huntingtin.
 22. The method according to claim 13, wherein said cancer is an invasive cancer and/or a cancer capable of metastasis.
 23. The method according to claim 13, wherein the cancer is selected from the group consisting of leukemia, lymphoma, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, liver cancer, pancreatic cancer, breast cancer, prostate cancer, testicular cancer and retinoblastoma.
 24. The compound according to claim 23, wherein the cancer is breast cancer or prostate cancer.
 25. The compound according to claim 13, wherein the subject is a human, preferably a human not affected with Huntington's disease. 